Seven mammalian isoforms of carbonic anhydrases are known. These enzymes possess carbon dioxide hydrating activity, believed to be important in regulation of intracellular pH. An inherited deficiency of carbonic anhydrase II (CAII) causes a syndrome of osteopetrosis, renal tubular acidosis, and cerebral calcification. CAIII is distinguished from the other isoforms by several characteristics, particularly by its low specific activity which is only ~1% that of CAII. Also, CAIII is not inhibited by acetozolamide, which is an excellent inhibitor of CAI and CAII. Finally, CAIII is unique among the other isoforms having a tyrosine phosphatase activity. However, even with the crystal structure of the protein available, the structural basis of the phosphatase is obscure. CAIII is remarkably high in muscle and adipocytes, constituting ~8 and 25% of the soluble protein content of these tissues. Despite numerous publications on the enzyme, the physiological function of CAIII remains unknown. Therefore, we are creating a knockout mouse to assist in defining the function of CAIII. Genomic DNA for mouse CAIII was cloned by the colony plaque hybridization method. Using a positive/negative type targeting vector, exons, 3, 4, and 5 of CAIII, were replaced by the bacterial neomycin (neo) resistence gene, driven by the phosphoglycerate kinase 1 promoter. The knockout vector was then electroporated into embryonic stem cells. G418 was used to select for cells containing the plasmid, and gancyclovir was used to select against cells in which the construct had integrated randomly. The resistant colonies were then grown to confluence, replica-plated, and genomic DNA was analyzed by a polymerase chain reaction (PCR) method. Eight correctly targeted clones were selected among the 118 double positive clones. These will be injected into host blastocysts, and the resultant chimeras will be selected for the desired CAIII knockout. Assessment of the development and growth of the homozygous knockout should illuminate the normal function of the enzyme.